A major function of the skin is to generate a protective barrier that shields us from our environment. This process is mediated by keratinocyte stem cells whose progeny stratify and differentiate, eventually forming an insoluble scaffold of dead cells and cross-linked proteinaceous material. Formation of this barrier is completed during embryonic development and is essential for life. Although the skin's role in barrier function is well-established, whether the hair follicle also plays a role in this process hs not been studied. In particular, the hair follicle infundibulum is a poorly characterized domain of cells that appears continuous with the skin epithelium. Situated at the mouth of the hair follicle, infundibular cells are multi-layered and exposed to the outer environment. These characteristics suggest both the need and capacity for infundibular cells to form a barrier. One of the missions of NIAMS is to support research that delves into the causes, treatment, and prevention of skin diseases. This proposal is concordant with that mission since our long-term objective is to characterize infundibular cells during embryonic development, adult homeostasis and disease perturbation. Indeed, a variety of skin disorders, including acne vulgaris, arises specifically at the infundibulum, and in more severe cases may cause rupture of this domain (and, presumably, disrupt barrier function), leading to inflammatory disease. This proposal seeks to shed light on the infundibulum, with an eye towards elucidating the mechanistic causes of acne. In Specific Aim 1, we will isolate both nascent and adult mouse infundibular cells, and characterize their gene expression patterns by RNA-Sequencing. We will also analyze in detail the movement of these cells as they form the differentiated, suprabasal layers of the infundibulum. Results from this opening Aim will improve our understanding of this domain, and provide tools and markers for use in the subsequent Aims. In Specific Aim 2, we will test the role of Notch signaling in the infundibulum by disrupting Notch pathway components specifically in the stem cells that give rise to the infundibulum in mice. Importantly, this will enable us to assess whether the hair follicle itself plays a role in barrier function, and may lead to the generation of improved mouse models of acne. In Specific Aim 3, we will examine whether Notch signaling and differentiation defects underlie acne formation in humans. This will be accomplished by analyzing a panel of markers, both for Notch signaling and the infundibulum, in patients prior to and after acne therapy. These translational studies will complement our basic experiments in mice, and will hopefully provide mechanistic insights into the causes of this disease